CN115025100A

CN115025100A - Application of effective components in preparing medicine for treating diabetic nephropathy

Info

Publication number: CN115025100A
Application number: CN202210741649.3A
Authority: CN
Inventors: 宋如珺; 韩赟
Original assignee: Suzhou Hospital of Traditional Chinese Medicine
Current assignee: Suzhou Hospital of Traditional Chinese Medicine
Priority date: 2022-06-28
Filing date: 2022-06-28
Publication date: 2022-09-09
Anticipated expiration: 2042-06-28
Also published as: CN115025100B

Abstract

The invention relates to application of active ingredients in preparation of a medicine for treating diabetic nephropathy, the active ingredients are Huihua vitexin B, the Huihua vitexin B can also be the only active ingredient, the active ingredients can be added with pharmaceutical excipients to prepare a preparation, and the preparation is one of tablets, capsules, granules and oral liquid.

Description

Application of effective components in preparing medicine for treating diabetic nephropathy

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of an active ingredient in preparation of a medicine for treating diabetic nephropathy, wherein the active ingredient is a Huihua vitexin B.

Background

Microvascular complications, particularly Diabetic Nephropathy (DN), are very common in patients with diabetes. DN is characterized by specific changes in kidney morphology and function. Among them, the early phase of DN is manifested by glomerular ultrafiltration, hypertrophy, microalbuminuria, thickening of basement membrane and expansion of the mesangial membrane. Late stage DN is characterized by a gradual decline in Glomerular Filtration Rate (GFR), massive proteinuria, decreased creatinine clearance, and glomerular and tubulointerstitial fibrosis. Mesangial cells are the main cell group in glomeruli and also the main source of glomerular matrix proteins, and dysfunction of mesangial cells is closely related to the development of diabetic nephropathy. In the course of progressing to overt renal disease, mesangial cells are lost by apoptosis, resulting in renal insufficiency. The development of DN involves a variety of pathogenic mechanisms, including hyperglycemia, hyperlipidemia, hypertension, proteinuria, oxidative stress, cytokines and genetic susceptibility among many other factors that can lead to the progression of diabetic nephropathy renal damage. Palmitic Acid (PA) is one of the most abundant saturated fatty acids in the Free Fatty Acid (FFA) component of the body, is a major component of the diet, and can also be synthesized de novo from carbohydrates. It is well known that mesangial cells, which are the main components of mesangial and the main targets of diabetes, are sensitive to lipotoxicity, acquire an activated phenotype in order to cope with diabetic factors such as hyperglycemia, oxidative stress and excess Free Fatty Acids (FFAs), and then undergo hypertrophy and proliferation or undergo apoptosis, resulting in renal dysfunction, i.e., lipotoxicity-induced mesangial apoptosis is associated with the occurrence of renal failure. Therefore, inhibition of lipotoxicity of mesangial cells is a potential therapeutic approach for the prevention and treatment of diabetic nephropathy.

Disclosure of Invention

The invention aims to: (1) the application of the effective components in preparing medicine for treating diabetic nephropathy is provided. (2) The effective component is Vitex agnus-casticin B. (3) The effective component of the vitexin B is added with pharmaceutic adjuvant to prepare the preparation.

In order to achieve the purpose, the invention provides the following technical scheme: the application of the effective component in preparing the medicine for treating diabetic nephropathy is characterized in that the effective component is the Huihua Vitexin B.

The effective component is applied to the preparation of the medicine for treating diabetic nephropathy, and the Huihua Vitexin B is the only effective component.

The effective components are added with pharmaceutical excipients to prepare the dosage form for the application in preparing the medicine for treating diabetic nephropathy.

The effective component is applied to preparing a medicine for treating diabetic nephropathy, and the dosage form is one of tablets, capsules, granules and oral liquid.

The use of the active ingredient of claim 1 in the manufacture of a medicament for the treatment of diabetic nephropathy, the active ingredient vitexin B being capable of increasing the viability of human mesangial cells.

The effective component of the active ingredient in the preparation of the medicine for treating diabetic nephropathy can increase the expression of the anti-apoptosis protein Bcl-2.

The effective component of the active ingredient is applied to preparing the medicine for treating diabetic nephropathy, and the active ingredient of the vitexin B can reduce the expression of a pro-apoptotic protein Bax and an apoptosis execution protein Cleved-caspase 3.

Vitexin B, CAS: 11027-63-7, formula: c ₂₂ H ₂₆ O ₁₁ Molecular weight: 466.439, available from Hubei Nocke pharmaceutical Co., Ltd.

Drawings

FIG. 1 shows the effect of vitexin B on the activity of human mesangial cells;

FIG. 2 shows the effect of vitexin B on human mesangial apoptotic proteins Bcl-2, Bax and caspase 3.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: taking 20g of the vitexin B, adding starch, and preparing into tablets.

Example 2: taking 20g of Huihua Vitexin B, adding dextrin, and preparing into granules.

Example 3: 20g of Huihua vitexin B is taken, added with starch and prepared into capsules.

Example 4: effect of Huihua Vitexin B on human mesangial cell viability

4.1 culture of human mesangial cells: taking out human mesangial cells (HMC cells) from liquid nitrogen, rapidly melting the human mesangial cells in a 37 ℃ water bath, sucking the completely melted cell suspension into a sterile EP tube by using a pipette gun, adding a proper amount of DMEM low-sugar culture medium, uniformly mixing, centrifuging at a low speed for 5 minutes, sucking and removing supernatant, adding 4ml of culture medium, blowing, uniformly mixing, transferring into a sterile culture bottle, and culturing in a 37 ℃ cell culture box. And observing the growth condition of the cells, and carrying out passage when the cell density reaches about 70-80%. Old medium was aspirated from the flask and washed 3 times with sterile PBS to remove residual serum. Adding 1ml of 0.25% pancreatin cell digestive juice, slightly shaking the culture flask to enable the pancreatin to fully contact the cells, putting the cells into an incubator to digest for 1min at 37 ℃, observing under a microscope, adding a complete cell culture medium containing serum to stop digestion when the cells begin to shrink and present round particles, blowing down the cells, collecting cell suspension, centrifuging for 5min at 1000rpm, removing supernatant, adding culture solution, slightly blowing up the cells until the cells are uniformly mixed, and subpackaging the uniformly mixed cell suspension into 2-3 culture flasks to continue culture.

4.2 grouping: the test group comprises a palmitic acid PA model group (600 mu mol/L), a normal control group and a treatment group (the palmitic acid PA and the Huihua vitexin B are respectively 0, 200, 400, 600, 8000 and 1000 mu mol/L, wherein the concentration of the palmitic acid PA is 600 mu mol/L.

4.3 determination of human mesangial Cell viability Using Cell Counting Kit-8(CCK-8) Kit: HMC cells in a logarithmic growth phase are taken, the cells are digested according to a method of 4.1, a cell counter is used for counting the cells, the HMC cells are inoculated in a 96-well culture plate at 8000 cells/well, the pore volume is 100ul, 200ul PBS is added in holes at the periphery of the plate, the cells are starved for 24 hours by a serum-free DMEM low-sugar culture medium when the cells grow to 70%, and the cells are synchronized and used for experiments. Experiments were performed in groups as described previously. After 24h of administration intervention, preparing CCK-8 detection solution (serum-free DMEM low-sugar medium: CCK-8 stock solution: 10: 1), discarding stock culture solution, adding CCK-8 detection solution, culturing 100ul of each well in a cell culture box for 1-4h, terminating the culture, and oscillating the 96-well plate on a shaking table at low speed in the dark for 10min to ensure that the color of the 96-well plate is uniform. The viability of each group of cells was calculated by placing the 96-well plate in a microplate reader, adjusting the wavelength to 450nm, and then measuring the OD value of each well. The cell survival rate was 100% (OD administration group-OD blank)/(OD control group-OD blank). Each set of 4 replicate wells was used in the experiment and replicated independently 3 times, see figure 1.

4.3 Experimental results: after the palmitic acid PA is subjected to the intervention treatment, the cell activity is remarkably reduced, which indicates that the molding is successful, and after the administration, the cell activity is gradually increased along with the increase of the concentration of the vitexin B. Therefore, the vitexin B can improve the activity of human glomerular mesangial cells.

Example 5: effect of Vitexin B on human mesangial cell apoptosis proteins Bcl-2, Bax and caspase3

5.1 human mesangial cell model establishment: after the human mesangial cells are recovered, the cells are cultured by a low-sugar DMEM medium, 10 percent of fetal calf serum and 1 percent of double antibodies (penicillin and streptomycin) are added, and the cells are cultured under the conditions of 37 ℃ and 5 percent of CO 2. Cells were passaged when grown to approximately 80% confluence. Cells were plated in 6-well plates, and when the cells grew to 70% confluence, the palmitic acid PA (600 μmol/L) intervention model was set up, and sugar-free DMEM medium was replaced and cultured for 6 hours. Adding HUIHUAMUJING B (final concentration of 40ng/ml) according to different culture conditions, culturing for 24 hr, and performing correlation detection. Dividing into normal culture group (without PA treatment), palmitic acid PA group, and fructus Viticis negundo glycoside B group (continuously culturing with fructus Viticis negundo glycoside B after palmitic acid PA), and performing related detection after continuously culturing for 24 hr.

5.2 Western blot procedure

1) Preparing glue: preparing separation gel and 5% concentrated gel with different concentrations according to the molecular weight of the target protein;

2) loading: after the gel is solidified, the comb is removed, the sample loading hole is washed by electrophoresis buffer solution, and each lane 10 is used for obtaining an L denatured protein sample;

3) electrophoresis: performing electrophoresis for 30min at constant voltage of 80V, adjusting the voltage to 120V when 72KDa appears, performing electrophoresis for about 50min, and stopping electrophoresis when bromothalein reaches the bottom of the separation gel;

4) cutting the glue: cutting out a target protein molecule adhesive tape according to the marker position;

5) film transfer: the target molecular adhesive tape is positioned at the negative electrode side, the PVDF film is positioned at the positive electrode side, the current is 300mA, and the film is rotated for 90min at 4 ℃;

6) and (3) sealing: after the membrane conversion is finished, the PVDF membrane is rinsed once by TBST, then is soaked in a confining liquid (5% skimmed milk + TBST), and is incubated for 1h at room temperature by shaking in a shaking table;

7) primary antibody incubation: diluting the antibody with a blocking solution, putting the blocked PVDF membrane into an antibody incubation box, adding 2mL of the antibody diluted in proportion into each strip, performing oscillation incubation at 4 ℃ by using a horizontal shaking table overnight;

8) and (3) secondary antibody incubation: the PVDF membrane is rewarmed for 30min at room temperature, and is washed 3 times and 510 min/time by TBST. The corresponding secondary antibody was diluted with blocking solution and incubated for 1h at room temperature.

9) Washing the membrane: washing the membrane with TBST for 3 times, 10min each time;

10) color development: developing with Immobilon Western chemiluminescence HRP Substrate kit from Millipore, and performing chemiluminescence with a chemiluminescence imager;

11) the grey values of the protein bands were analyzed by Image J for statistical analysis.

5.3 results: WB detection shows that compared with Control group, under the action of PA, the content of anti-apoptotic protein Bcl-2 in HMC cells is reduced, the content of pro-apoptotic protein Bax and apoptosis executive protein Cleved-caspase3 is increased, PA induces apoptosis of human mesangial cells, and after the treatment of vitexin B, the content of anti-apoptotic protein Bcl-2 is increased, and the content of pro-apoptotic protein Bax and apoptosis executive protein Cleved-caspase3 is reduced, which shows that microglia treated by vitexin B can reduce apoptosis of human mesangial cells after PA intervenes, protect renal function and further protect cells.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. The application of the effective component in preparing the medicine for treating diabetic nephropathy is characterized in that the effective component is the vitexin B.

2. The use of the active ingredient as claimed in claim 1 for the preparation of a medicament for the treatment of diabetic nephropathy, wherein vitexin B is the only active ingredient.

3. The use of the active ingredient of claim 1 in the preparation of a medicament for the treatment of diabetic nephropathy, wherein the active ingredient is added with pharmaceutical excipients and formulated into a dosage form.

4. The use of the active ingredient of claim 3 in the preparation of a medicament for the treatment of diabetic nephropathy, wherein the dosage form is one of tablets, capsules, granules and oral liquid.

5. The use of the active ingredient of claim 1 for the preparation of a medicament for the treatment of diabetic nephropathy, wherein the active ingredient vitexin B is capable of increasing the viability of human mesangial cells.

6. The use of the active ingredient of claim 1 for the preparation of a medicament for the treatment of diabetic nephropathy, wherein the active ingredient vitexin B increases the expression of the anti-apoptotic protein Bcl-2.

7. The use of the active ingredient of claim 1 for the preparation of a medicament for the treatment of diabetic nephropathy, wherein the active ingredient of vitexin B decreases the expression of pro-apoptotic protein Bax and executive apoptotic protein Cleved-caspase 3.